mRNA Expression Profiling from Extracellular Vesicles (EVs): Generating a Rapid Diagnostic for Stroke

项目总结/摘要 中风是一种脑部疾病，其中一部分失去血液供应，受影响的区域迅速发展 如果血流不能及时恢复就会死亡急性缺血性卒中（AIS）后恢复血流的治疗 如果在中风事件发生后<4.5小时给药，则有效。但是，部分原因是缺乏体外 需要在主治医院进行AIS诊断测试、成像（CT -临床灵敏度~26%）才能诊断。 因此，<7%的AIS患者接受治疗。因此，迫切需要制定战略， 在接近真实的时间内诊断中风，这可能允许即时检测（POCT）。一种方法是 一种使用标记物的外周血测试，该标记物对中风引起的大脑变化做出快速反应。的 拟议的项目将开发利用外周血标记物诊断中风的创新技术 在接近真实的时间内（采样至回答时间约为31分钟），LOD约为0.03 ng（总RNA）。研究小组发现，从白色血细胞中获得的mRNA表达的改变 亚群可用于中风诊断，并在中风事件后迅速出现在外周血中。 根据先前的R 01，发现CD 15+和CD 8+白细胞是卒中的主要来源。 相关的mRNA生物标志物。该项目旨在实现创新的射流盒的开发， 用于测量来源于CD 15和CD 8的中风相关RNA标志物的相关测定 表达细胞外囊泡（EV）。EV作为中风相关RNA生物标志物来源的效用是基于 它们在血浆中的快速出现和高丰度，可能提供更快的中风诊断 与它们所来源的细胞相比。该项目将发现具有高度临床应用价值的EV-RNA标志物， 诊断缺血性和出血性卒中的敏感性和特异性（>80%）。的 盒，其由通过复制由塑料制成的任务特定模块组成（即，注塑成型） 连接到一个流体主板，将使用来自这个项目的EV-RNA标记。等离子体将用于 作为输入，从该输入，将使用专门设计的免疫调节剂发生CD 8和CD 15 EV的表面亲和选择。 module.在EV经由可光裂解的接头（使用蓝光切割）从捕获表面释放之后， LED），检测盒将使用无标记读出策略量化选择的EV数量。流体 检测盒还包括一个混合刻度（nm → µm）模块，用于电子读取中风EV-RNA的拷贝数 以高度多路复用的方式（>24个靶标）检测标记物。该模块将由在纳米结构中制成的平面内纳米孔组成。 塑料通过纳米注塑成型，并可以识别RNA使用不同的寡核苷酸引物对查询 使用固相连接酶检测反应（spLDR）检测特异性RNA。单分子读出将允许高 分析灵敏度，以观察EV-RNA标记拷贝数的细微变化。该墨盒的实用性将 在临床环境中进行评估。项目的成功得益于强大的多学科团队， 在开发中风标志物和中风诊断平台方面有着富有成效的合作记录。

Project Summary/Abstract Stroke is a disorder of the brain by which a part loses its blood supply and the affected region rapidly progresses to death if blood flow is not restored in time. Treatments to restore blood flow after acute ischemic stroke (AIS) are effective if administered <4.5 h from the onset of the stroke event. But, due in part to the lack of an in vitro diagnostic test for AIS, imaging (CT – clinical sensitivity ~26%) at the attending hospital is required for diagnosis. As a result, <7% of AIS patients receive treatment. Therefore, a critical need exists to develop strategies for diagnosing stroke in near real time that potentially can allow for point-of-care testing (POCT). One approach is a peripheral blood test using markers that quickly respond to changes in the brain induced by stroke. The proposed project will develop innovative technologies that uses peripheral blood markers for diagnosing stroke syndromes (AIS and hemorrhagic stroke) in near real time (~31 min for sample-to-answer) with an LOD of ~0.03 ng (total RNA). The research team has found that alterations in mRNA expression secured from white blood cell subsets can be used for stroke diagnosis and appear rapidly in peripheral blood following a stroke event. Resulting from a prior R01, CD15+ and CD8+ leukocytes were discovered as a predominant source of stroke- related mRNA biomarkers. This project seeks to realize the development of an innovative fluidic cartridge and the associated assay for the measurement of stroke-related RNA markers sourced from CD15 and CD8 expressing extracellular vesicles (EVs). The utility of EVs as a source of stroke-related RNA biomarkers is based on their rapid appearance and high abundance in plasma, potentially providing even faster stroke diagnosis compared to the cells from which they originate. This project will discover EV-RNA markers with high clinical sensitivity and specificity (>80%) for diagnosing ischemic and hemorrhagic stroke in <3 h from stroke onset. The cartridge, which consists of task-specific modules made from plastics via replication (i.e., injection molding) connected to a fluidic motherboard, will use the EV-RNA markers emanating from this project. Plasma will serve as the input from which surface-affinity selection of CD8 and CD15 EVs will occur using a specifically designed module. Following EV release from the capture surface via a photocleavable linker (cleaved using a blue-light LED), the cartridge will quantify the number of EVs selected using a label-free readout strategy. The fluidic cartridge also consists of a mixed-scale (nm → µm) module to read electrically copy numbers of stroke EV-RNA markers in a highly multiplexed fashion (>24 targets). This module will consist of in-plane nanopores made in a plastic via nano-injection molding and can identify RNAs using a distinct oligonucleotide primer pair querying a specific RNA using a solid-phase ligase detection reaction (spLDR). Single-molecule readout will allow for high analytical sensitivity to observe subtle changes in EV-RNA marker copy numbers. The utility of this cartridge will be evaluated in a clinical setting. Success of the project is leveraged by a strong multidisciplinary team, whom have a productive record of collaboration in developing stroke markers and diagnostic platforms for stroke.